Abstract
The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: bla KPC, bla NDM, bla OXA-48-like, and bla IMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for bla KPC, bla NDM, and bla OXA-48-like) or 1000 fg/reaction (for bla IMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 bla KPC, 69 bla NDM, 3 bla OXA-48-like, and 1 bla IMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.
Highlights
Enterobacterales are conditionally pathogenic bacteria that cause serious hospital-acquired infections (Feil, 2017)
Seven other common pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis were used to validate the specificity of the Recombinase polymerase amplification (RPA)-lateral flow strips (LFS) method
The blaKPC, blaNDM, blaOXA-48-like, and blaIMP genes were used as target sequences, and five pairs of primer sets were designed for each gene targeting to the highly conserved area
Summary
Enterobacterales are conditionally pathogenic bacteria that cause serious hospital-acquired infections (Feil, 2017). The spread of carbapenemase-producing Enterobacterales (CPE) has become a major global public health threat. Carbapenems have traditionally been used to treat infections caused by broad-spectrum betalactamase-producing Escherichia coli and Klebsiella pneumoniae and are still considered antibiotics to be used as a last resort (Nordmann et al, 2012; van Duin and Doi, 2017; Rochford et al, 2018; Lin et al, 2020). Carbapenemase-producing enzymes in these bacteria, which are capable of hydrolyzing all carbapenems, cephalosporins, and beta-lactams are the main cause of resistance to carbapenem antibiotics (Khan et al, 2017; Segagni Lusignani et al, 2020). Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-b-lactamase (NDM), oxacillinase (OXA-48like), and imipenemase (IMP) are the most prevalent carbapenemases in CPE (Han et al, 2020)
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